Correcting an assay image of an array of signals generated from a multiplexed hybridization-mediated assay

ABSTRACT

Described are methods of assay design and assay image correction, useful for multiplexed genetic screening for mutations and polymorphisms, including CF-related mutants and polymorphs, using an array of probe pairs (in one aspect, where one member is complementary to a particular mutant or polymorphic allele and the other member is complementary to a corresponding wild type allele), with probes bound to encoded particles (e.g., beads) wherein the encoding allows identification of the attached probe. The methods relate to avoiding cross-hybridization by selection of probes and amplicons, as well as separation of reactions of certain probes and amplicons where a homology threshold is exceeded. Methods of correcting a fluorescent image using a background map, where the particles also contain an optical encoding system, are also disclosed.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional No. 60/470,806,filed May 15, 2003.

BACKGROUND

The standard method of genomic analysis for mutations and polymorphisms,including for CF, is the “dot-blot” method. Samples including targetstrands are spotted onto a nitrocellulose support, and then contactedwith labeled probes complementary to the mutations or polymorphicregions. The labels allow detection of probe hybridization toimmobilized complementary target sequences, as unbound labeled probesare removed by washing. In another method—a “reverse dot-blot format”—anarray of oligonucleotide probes is bound to a solid support, and thencontacted with a sample including target sequences of interest. See,e.g., U.S. Pat. No. 5,837,832.

Both methods of assaying mutations or polymorphisms have significantdisadvantages. The dot-blot method is itself labor-intensive. It canalso yield erroneous results due to the inaccurate reading of assaysignals, usually done by autoradiography, which adds further labor, asthe probes must be frequently re-labeled. The method described in U.S.Pat. No. 5,837,832 involves a complex and costly on chip synthesis of anarray of oligonucleotides, an approach which is better-suited forlarge-scale genomic analysis and is neither practical nor cost-effectivefor diagnostic applications requiring only a limited but changing numberof probes.

An assay method suitable for multiplexed analysis which avoids many ofthe problems associated with the above methods involves use of randomencoded arrays of microparticles, where the encoding indicates theidentity of an oligonucleotide probe molecule bound thereto. See. U.S.patent application Ser. No. 10/204,799: “Multianalyte Molecular AnalysisUsing Application-Specific Random Particle Arrays.” The bead array iscontacted with labeled amplicons, generated from a patient sample, andthe labels are then detected (if the labels are fluorescent, thedetection can be with optical means) and the bound amplicons areidentified by decoding of the array.

In a multiplexed hybridization assay, cross-hybridization amongmis-matched, but closely homologous, probes and amplicons can generatefalse positive signals. Thus, the assay should be designed to minimizesuch effects. A number of mutations and polymorphisms are significantonly if they are homozygous, and therefore, to be useful in such cases,the assay must be capable of discriminating heterozygotes fromhomozygotes. Also, in determining the assay results, where both theencoding method for the beads and the determination of assay results iswith optically detectable means, the encoding on the beads can causespectral leakage, which can be affect the assay signal discrimination. Amethod of correcting for such spectral leakage is also needed.

Cystic fibrosis (“CF”) is one of the most common recessive disorders inCaucasians, with an occurrence of 1 in 2000 live births in the UnitedStates. Mutations in the cystic fibrosis (CF) transmembrane conductanceregulator (CFTR) gene are associated with the disease. The number of.CFTR mutations is growing continuously and rapidly, and more than 1,000mutations have been detected to date. See Kulczycki L. L., et al.(2003), Am J Med Genet 116:262-67. Population studies have indicatedthat the most common CF mutation, a deletion of the 3 nucleotides thatencode phenylalanine at position 508 of the CFTR amino acid sequence(designated ΔF508), is associated with approximately 70% of the cases ofcystic fibrosis. This mutation results in the failure of an epithelialcell chloride channel to respond to cAMP (Frizzell R. A. et al. (1986)Science 233:558-560; Welsh, M. J. (1986) Science 232:1648-1650.; Li, M.et al. (1988) Nature 331:358-360; Quinton, P. M. (1989) Clin. Chem.35:726-730). In airway cells, this leads to an imbalance in ion andfluid transport. It is widely believed that this causes abnormal mucussecretion observed in CF patients, and ultimately results in pulmonaryinfection and epithelial cell damage. A number of mutations areassociated with CF, and researchers continue to reveal new mutationsassociated with the disease. The American College of Medical Genetics(“ACMG”) has recommended a panel of 25 of the most common CF-associatedmutations in the general population, especially those in AshkenaziJewish and African-American populations. A multiplexed hybridizationassay for CF-associated mutations in the general population would testfor this panel.

SUMMARY

Described are practical and cost-effective methods of assay design andassay image correction, useful for multiplexed genetic screening formutations and polymorphisms, including CF-related mutants andpolymorphs, using an array of probe pairs (in one aspect, where onemember is complementary to a particular mutant or polymorphic allele andthe other member is complementary to a corresponding wild type allele),with probes bound to encoded particles (e.g., beads) wherein theencoding allows identification of the attached probe. The design methodsdisclosed herein were used to design an assay for CF-related mutationsby hybridization-mediated multiplexed analysis, and were extensivelyvalidated in many patient samples, and demonstrated to be capable ofidentifying the most common mutations, including mutations in exons 3,4, 5, 7, 9, 10, 11, 13, 14b, 16, 18, 19, 20, 21 and introns 8, 12, 19 ofthe CFTR gene.

Before hybridization, the region of interest in the genomic sample isamplified with two primers, one for each strand in the region ofinterest. Of the two strands generated in the PCR amplification step,one is arbitrarily designated herein as “sense” and one as “anti-sense.”In certain instances, it is desirable to select, for subsequent mutationanalysis by hybridization, either the sense target strand (to behybridized to sense probes) or the anti-sense target strand—to behybridized to anti-sense probes. Strand selection is accomplished, forexample, by post-PCR digestion of a phosphorylated strand. Inparticular, strand switching is desirable whenever probe-targetcombinations (e.g., sense-probe/sense target hybridization) involving astable mismatch configuration, such as a G-T base pairing, can beavoided.

Also disclosed are methods of selecting probes and amplicons for geneticscreening for mutations and polymorphisms. The method of selectingprobes and amplicons involves the following steps:

-   providing a family of single-stranded MP amplicons in which one    strand is designated sense and the complementary strand is    designated anti-sense, said MP amplicons including amplified    segments of the genome on which said genetic mutations or    polymorphisms are located;-   selecting complementary MP probes for each member of said family of    MP amplicons;-   examining the degree of homology between either the complementary MP    probes or between the family of MP amplicons;-   dividing said MP probes into one or more probe sets, and dividing    said MP amplicons into sets such that the members of each amplicon    set are complementary to the members of one probe set, said division    based on avoiding homology greater than an acceptance level between    probes in the same set or between MP amplicons in the same set;-   performing for each said set of amplicons in turn, the following    steps for each MP amplicon in said set, in succession:-   (a) (i) determining whether, upon contacting a sense MP amplicon    with a probe set which includes a complementary MP probe to said    sense amplicon, the degree of cross-hybridization of said sense MP    amplicon with other MP probes in the probe set will exceed an    acceptance level; and, if not:-   (a)(ii) retaining said sense MP amplicon in the amplicon set and the    complementary MP probe in the probe set, and repeating step (a) (i)    for another MP amplicon in said family;-   (b)(i) but if said degree of cross-hybridization does exceed said    acceptance level: replacing, in the probe set, the cross-hybridizing    MP probe with the complementary anti-sense MP probe, and replacing,    in the amplicon set, the complementary sense MP amplicon with the    anti-sense MP amplicon complementary to said anti-sense MP probe,    and-   (b)(ii) repeating step (a)(i) and if the degree of    cross-hybridization is within the acceptance level: retaining said    anti-sense MP probe and corresponding complementary anti-sense MP    amplicon in their respective sets and repeating step (a)(i);-   (b)(iii) but if the degree of cross-hybridization exceeds the    acceptance level after repeating step (a)(i): determining whether,    upon contacting said anti-sense MP amplicon with the MP probes in    any other set, the degree of cross-hybridization is within the    acceptance level, and if so, placing the anti-sense MP probe    complementary to said anti-sense MP amplicon into said set and    placing said anti-sense MP amplicon into the set of complementary    anti-sense MP amplicons; but if the degree of cross-hybridization    exceeds the acceptance level following such determination for each    existing probe set, reverting to the original sense MP probe and    complementary sense MP amplicon and placing said sense MP probe and    said complementary sense MP amplicon each into a new set, and-   (c) repeating steps (a) to (c) for another sense MP amplicon in said    family.

Also disclosed is a method for design of pairs of probes (with a memberrespectively complementary to a mutant and a wild type amplicon) forhybridization to labeled amplicons generated by amplification of samplesand wild type controls. For each anticipated variant, probes areprovided in pairs, with one member complementary to the wild typesequence and the other to the variant sequence, the two sequences oftendiffering by only one nucleotide. One method to enhance the reliabilityof hybridization-mediated multiplexed analysis of polymorphisms (hMAP)is to determine the ratio of the signals generated by the capture of thetarget matched and mismatched probes and to set relative ranges ofvalues indicative of normal and heterozygous or homozygous variants.

The method set forth above for selecting probes and amplicons forgenetic screening for mutations and polymorphisms, can be included aspart of a method to select probe pairs (wild-type and variant), byincluding the following steps in the afore-described method:

-   providing a family of single-stranded WT amplicons in which one    strand is designated sense and the complementary strand is    designated anti-sense, said family representing respective amplified    segments of a wild type genome which corresponds to each of the    amplified segments of the genome which was amplified when producing    the family of MP amplicons;-   providing and selecting a sense or anti-sense WT probe so as to have    both a sense WT probe and a corresponding sense MP probe in the same    probe set or, or an anti-sense WT probe and a corresponding    anti-sense MP probe in the same probe set;-   determining: (i) whether the degree of cross-hybridization between a    MP amplicon and a corresponding WT probe in a probe set, and between    a WT amplicon and a corresponding MP probe in a probe set, will    exceed the acceptance level and, if so, (ii) determining whether    said degree of cross-hybridization will fall within the acceptance    level if the selected sense or anti-sense MP and WT probes are    replaced with the complementary WT and MP probes; and if so, (iii)    determining whether said complementary WT and MP probes will exceed    the acceptance level for cross-hybridization with amplicons    complementary to other members of the same probe set, and if    so, (iv) determining whether placing the complementary WT and MP    probes into another probe set will exceed the acceptance level for    cross-hybridization with amplicons complementary to other members of    the same probe set, and if not: retaining the complementary WT and    MP probes in said probe set; but if so, (v) repeating step (iv) for    each existing probe set, and if said acceptance level is exceeded    for each existing probe set, placing the complementary WT and MP    probes into a new set and placing the complementary WT and MP    amplicons into a corresponding new set.

Cross-hybridization is a concern in any assay involving multiplexedhybridization, and methods to avoid its deleterious effects on assayresults are included herein. One method to correct forcross-hybridization in an array format, is to set a series oftemperature increments, selected such that at each temperature,probe-target complexes containing particular mismatch configurationswill denature, while those containing matched (“complementary”) basepair configurations will remain intact. The signals generated bycaptured labeled strands hybridized to probes in the array are thenmonitored and recorded at each temperature set point. Analysis of theevolution of differential signals as a function of temperature allowscorrection for each mismatch expected to become unstable above a certain“melting” temperature. After all set points for all mismatches aredetermined, data gathered at lower temperatures can be corrected for allmismatches.

In another aspect, because the assay method herein relies on encodedbeads to identify the probe(s) attached thereto, and the encoding in oneembodiment is by way of dye staining, the assay signals are oftenproduced by using fluorescent labels and removing backgroundcontributions. Specifically, a method of correcting the assay image isdisclosed. That is, within the spectral band selected for the recordingof the assay image, the recorded set of optical signatures produced bytarget capture to bead-displayed probes in the course of the assay arecorrected for the effects of “spectral leakage” (a source of spuriouscontributions to the assay image from the residual transmission) ofintensity emitted by bead-encoding dyes of lower wavelength. An assaydesign is provided herein in which a negative control bead is includedin the random encoded array for each type of encoded bead that producesunacceptably large spectral leakage, for example, for beads containingdifferent amounts of specific encoding dyes.

In the examples described herein, negative control beads display an18-mer C polynucleotide in order to serve a secondary purpose, i.e., topermit correction of assay images for the effects of non-specificadsorption. Preferably, the background correction is performed byconstructing a background map based on the random locations of each typeof negative control bead, where each such type of negative control beadis included in the array at a pre-selected abundance. For each type ofnegative control bead within the array, a background map is generated bylocating the centroids of the beads of that type, constructing theassociated Voronoi tessellation by standard methods (as illustrated inFIG. 3; see, e.g., Seul, O'Gorman & Sammon, “Practical Algorithms forImage Analysis,” Cambridge University Press, 2000; at page 222;incorporated by reference) and then filling each polygon which includesa bead with the intensity of such bead to produce a map (see, e.g., themap shown in FIG. 3). Optionally, standard filtering operations may beapplied to smooth the map; that is, to average out effects fromneighboring pixels. See, e.g., Seul, O'Gorman & Sammon, “PracticalAlgorithms for Image Analysis,” Cambridge University Press, 2000 fordescription of a filter).

Such a map represents a finite sample of the entire backgroundcontributions to the assay image in a manner that accounts for certainnon-linear optical effects associated with arrays composed of refractivebeads, which effects are especially pronounced when the beads are placedinto mechanical traps on a substrate surface. In addition, backgroundmaps will indicate non-uniformities in the background which may arise,for example, from non-uniform illumination or non-uniform distributionof target or analyte placed in contact with the bead array. Maps fornegative control beads of different types, i.e., containing differentamounts of encoding dyes and producing different degrees of spectralleakage, may be normalized to the same mean intensity and superimposedto increase the sampling rate.

The assay image may be corrected as follows by employing the backgroundmap. In certain instances, the map is simply subtracted from the assayimage to produce a corrected assay image. In other embodiments, thebackground can be combined with a “flat fielding” step (See, e.g., Seul,O'Gorman & Sammon, “Practical Algorithms for Image Analysis,” CambridgeUniversity Press, 2000). In this procedure, the constant (i.e., thespatially non-varying) portions of the background map and assay imageare subtracted, and the corrected assay image is divided by thecorrected background map to obtain a “flat fielded” intensity map.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of hybridization of 29 different CFTRmutations; where the smaller open bars represent mutant hybridization,and where hybridization to the “normal” is represented by the largerblack bars (e.g., EX-10 has a high degree of mutant hybridization).

FIG. 2 shows the results of hybridization of 29 CFTR mutations, with themutations being different from those shown in FIG. 1.

FIG. 3 shows a background map of negative control carriers forcorrecting array images.

DETAILED DESCRIPTION

Provided herein are methods for hybridization-mediated multiplexedanalysis of polymorphisms (hMAP) of a designated set of designatedmutations in he Cystic Fibrosis Transmembrane Conductance Regulator(CFTR) gene.

Probes used in the detection of mutations in a target sequence hybridizewith high affinity to amplicons generated from designated target sites,when the entire amplicon, or a subsequence thereof, is fullycomplementary (“matched”) to that of the probe, but hybridize with alower affinity to amplicons which have no fully complementary portions(“mismatched”). Generally, the probes of the invention should besufficiently long to avoid annealing to unrelated DNA target sequences.In certain embodiments, the length of the probe may be about 10 to 50bases, or preferably about 15 to 25 bases, and more preferably 18 to 20bases.

Probes are attached, via their respective 5′ termini, using linkermoieties through methods well known in the art, to encodedmicroparticles (“beads”) having a chemically or physicallydistinguishable characteristic uniquely identifying the attached probe.Probes are designed to capture target sequences of interest contained ina solution contacting the beads. Hybridization of target to the probedisplayed on a particular bead produces an optically detectablesignature. The optical signature of each participating bead uniquelycorresponds to the probe displayed on that bead. Prior to, or subsequentto the hybridization step, one may determine the identity of the probesby way of particle identification and detection, e.g., by decoding orusing multicolor fluorescence microscopy.

The composition of the beads includes, but is not limited to, plastics,ceramics, glass, polystyrene, methylstyrene, acrylic polymers,paramagnetic materials, thoria sol, carbon graphite, titanium dioxide,latex or cross-linked dextrans such as sepharose, cellulose, nylon,cross-linked micelles and Teflon. See “Microsphere Detection Guide” fromBangs Laboratories, Fishers Ind. The particles need not be spherical andmay be porous. The bead sizes may range from nanometers (e.g., 100 nm)to millimeters (e.g., 1 mm), with beads from about 0.2 micron to about200 microns being preferred, more preferably from about 0.5 to about 5micron being particularly preferred.

In certain embodiments, beads may be arranged in a planar array on asubstrate prior to the hybridization step. Beads also may be assembledon a planar substrate to facilitate imaging subsequent to thehybridization step. The process and system described herein provide ahigh throughput assay format permitting the instant imaging of an entirearray of beads and the simultaneous genetic analysis of multiple patientsamples.

The array of beads may be a randomly encoded array, that is, the codeassociated with each bead, placed during assembly into a position withinthe array that is not known a priori, indicates the identity ofoligonucleotide probes attached to said beads. Random encoded arrays maybe formed according to the methods and processes disclosed inInternational Application No. PCT/US01/20179, incorporated herein byreference.

The bead array may be prepared by employing separate batch processes toproduce application-specific substrates (e.g., chip at the wafer scale)to produce beads that are chemically encoded and attached tooligonucleotide probes (e.g., at the scale of about 10⁸ beads/100 μlsuspension). These beads are combined with a substrate (e.g., siliconchip) and assembled to form dense arrays on a designated area on thesubstrate. In certain embodiments, the bead array contains 4000 of 3.2μm beads has a dimension of 300 μm by 300 μm. With different size beads,the density will vary. Multiple bead arrays can also be formedsimultaneously in discrete fluid compartments maintained on the samechip. Such methods are disclosed in U.S. application Ser. No.10/192,352, entitled: ““Arrays of Microparticles and Methods ofPreparation Thereof,” which is incorporated herein by reference. Beadarrays may be formed by the methods collectively referred to as“LEAPS™”, as described in U.S. Pat. Nos. 6,251,691, 6,514,771; 6,468,811all of which are also incorporated herein by reference.

Substrates (e.g., chips) used in the present invention may be a planarelectrode patterned in accordance with the interfacial patterningmethods of LEAPS by, e.g., patterned growth of oxide or other dielectricmaterials to create a desired configuration of impedance gradients inthe presence of an applied AC electric field. Patterns may be designedso as to produce a desired configuration of AC field-induced fluid flowand corresponding particle transport. Substrates may be patterned on awafer scale by invoking semiconductor processing technology. Inaddition, substrates may be compartmentalized by depositing a thin filmof a UV-patternable, optically transparent polymer to affix to thesubstrate a desired layout of fluidic conduits and compartments toconfine fluid in one or several discrete compartments, therebyaccommodating multiple samples on a given substrate.

The bead arrays may be prepared by providing a first planar electrodethat is in substantially parallel to a second planar electrode(“sandwich” configuration) with the two electrodes being separated by agap and containing a polarizable liquid medium, such as an electrolytesolution. The surface or the interior of the second planar electrode ispatterned with the interfacial patterning method. The beads areintroduced into the gap. When an AC voltage is applied to the gap, thebeads form a random encoded array on the second electrode (e.g.,“chip”). And, also using LEAPS, an array of beads may be formed on alight-sensitive electrode (“chip”). Preferably, the sandwichconfiguration described above is also used with a planar light sensitiveelectrode and another planar electrode. Once again, the two electrodesare separated by a gap and contain an electrolyte solution. Thefunctionalized and encoded beads are introduced into the gap. Uponapplication of an AC voltage in combination with a light, the beads forman array on the light-sensitive electrode.

In certain embodiments, beads may be associated with a chemically oroptically distinguishable characteristic. This may be provided, forexample, by staining beads with sets of optically distinguishable tags,such as those containing one or more fluorophore or chromophore dyesspectrally distinguishable by excitation wavelength, emissionwavelength, excited-state lifetime or emission intensity. The opticallydistinguishable tags made be used to stain beads in specified ratios, asdisclosed, for example, in Fulwyler, U.S. Pat. No. 4,717,655 (Jan. 5,1988). Staining may also be accomplished by swelling of particles inaccordance with methods known to those skilled in the art, (Molday,Dreyer, Rembaum & Yen, J. Mol Biol 64, 75-88 (1975); L. Bangs, “Uniformlatex Particles, Seragen Diagnostics, 1984). For example, up to twelvetypes of beads were encoded by swelling and bulk staining with twocolors, each individually in four intensity levels, and mixed in fournominal molar ratios. Alternatively, the methods of combinatorial colorencoding described in International Application No. PCT/US 98/10719,incorporated herein by reference, can be used to endow the bead arrayswith optically distinguishable tags. In addition to chemical encoding,beads may also be rendered magnetic by the processes described ininternational Application No. WO 01/098765.

In addition to chemical encoding of the dyes, the beads having certainoligonucleotide primers may be spatially separated (“spatial encoding”),such that the location of the beads provide certain information as tothe identity of the beads placed therein. Spatial encoding, for example,can be accomplished within a single fluid phase in the course of arrayassembly by invoking LEAPS to assemble planar bead arrays in any desiredconfiguration in response to alternating electric fields and/or inaccordance with patterns of light projected onto the substrate.

LEAPS creates lateral gradients in the impedance of the interfacebetween silicon chip and solution to modulate the electrohydrodynamicforces that mediate array assembly. Electrical requirements are modest:low AC voltages of typically less than 10V_(pp) are applied across afluid gap of typically 100 μm between two planar electrodes. Thisassembly process is rapid and it is optically programmable: arrayscontaining thousands of beads are formed within seconds under electricfield. The formation of multiple subarrays, can also occur in multiplefluid phases maintained on a compartmentalized chip surface.

Subsequent to the formation of an array, the array may be immobilized.For example, the bead arrays may be immobilized, for example, byapplication of a DC voltage to produce random encoded arrays. The DCvoltage, set to typically 5-7 V (for beads in the range of 2-6 μm andfor a gap size of 100-150 μm) and applied for <30 s in “reverse bias”configuration so that an n-doped silicon substrate would form the anode,causes the array to be compressed to an extent facilitating contactbetween adjacent beads within the array and simultaneously causes beadsto be moved toward the region of high electric field in immediateproximity of the electrode surface. Once in sufficiently closeproximity, beads are anchored by van der Waals forces mediating physicaladsorption. This adsorption process is facilitated by providing on thebead surface a population of “tethers” extending from the bead surface;polylysine and streptavidin have been used for this purpose.

In certain embodiments, the particle arrays may be immobilized bychemical means, e.g, by forming a composite gel-particle film. In oneexemplary method for forming such gel-composite particle films, asuspension of microparticles is provided which also contain allingredients for subsequent in-situ gel formation, namely monomer,crosslinker and initiator. The particles are assembled into a planarassembly on a substrate by application of LEAPS, e.g., AC voltages of1-20 V_(p-p) in a frequency range from 100's of hertz to severalkilohertz are applied between the electrodes across the fluid gap.Following array assembly, and in the presence of the applied AC voltage,polymerization of the fluid phase is triggered by thermally heating thecell ˜40-45° C. using an infra-red (IR) lamp or photometrically using amercury lamp source, to effectively entrap the particle array within agel. Gels may be composed of a mixture of acrylamide and bisacrylamideof varying monomer concentrations from 20% to 5%(acrylamide:bisacrylamide=37.5:1, molar ratio), or any other lowviscosity water soluble monomer or monomer mixture may be used as well.Chemically immobilized functionalized microparticle arrays prepared bythis process may be used for a variety of bioassays, e.g., ligandreceptor binding assays.

In one example, thermal hydrogels are formed using azodiisobutyramidinedihydrochloride as a thermal initiator at a low concentration ensuringthat the overall ionic strength of the polymerization mixture falls inthe range of ˜0.1 mM to 1.0 mM. The initiator used for the UVpolymerization is Irgacure 2959®(2-Hydroxy-4′-hydroxyethoxy-2-methylpropiophenone, Ciba Geigy,Tarrytown, N.Y.). The initiator is added to the monomer to give a 1.5%by weight solution.

In certain embodiments, the particle arrays may be immobilized bymechanical means. For example, an array of microwells may be produced bystandard semiconductor processing methods in the low impedance regionsof the silicon substrate. The particle arrays may be formed using suchstructures by, e.g., utilizing LEAPS mediated hydrodynamic andponderomotive forces are utilized to transport and accumulate particleson the hole arrays. The AC field is then switched off and particles aretrapped into microwells and thus mechanically confined. Excess beads areremoved leaving behind a geometrically ordered random bead array on thesubstrate surface.

Substrates (e.g., chips) can be placed in one or more enclosedcompartment, permitting interconnection. Reactions can also be performedin an open compartment format similar to microtiter plates. Reagents maybe pipetted on top of the chip by robotic liquid handling equipment, andmultiple samples may be processed simultaneously. Such a formataccommodates standard sample processing and liquid handling for existingmicrotiter plate format and integrates sample processing and arraydetection.

Encoded beads can also be assembled, but not in an array, on thesubstrate surface: For example, by spotting bead suspensions intomultiple regions of the substrate and allowing beads to settle undergravity, assemblies of beads can be formed on the substrate. In contrastto the bead arrays formed by LEAPS, these assemblies generally assumedisordered configurations of low-density or non-planar configurationsinvolving stacking or clumping of beads thereby preventing imaging ofaffected beads. However, the combination of spatial and color encodingattained by spotting mixtures of chemically encoded beads into amultiplicity of discrete positions on the substrate still allowsmultiplexing.

In certain embodiments, a comparison of an assay with a decoded image ofthe array can be used to reveal chemically or physically distinguishablecharacteristics, and the elongation of probes. This comparison can beachieved by using, for example, an optical microscope with an imagingdetector and computerized image capture and analysis equipment. Theassay image of the array is taken to detect the optical signature thatindicates the probe elongation. The decoded image may be taken todetermine the chemically and/or physically distinguishablecharacteristics that uniquely identify the probe displayed on the beadsurface. In this way, the identity of the probe on each particle in thearray may be identified by a distinguishable characteristic.

Image analysis algorithms may be used in analyzing the data obtainedfrom the decoding and the assay images. These algorithms may be used toobtain quantitative data for each bead within an array. The analysissoftware automatically locates bead centers using a bright-field imageof the array as a template, groups beads according to type, assignsquantitative intensities to individual beads, rejects “blemishes” suchas those produced by “matrix” materials of irregular shape in serumsamples, analyzes background intensity statistics and evaluates thebackground-corrected mean intensities for all bead types along with thecorresponding variances. Examples of such algorithms are set forth inInternational Application No. WO 01/098765.

The probe hybridization may be indicated by a change in the opticalsignature, e.g., of the beads associated with the probes. This can bedone using labeling methods well known in the art, including direct andindirect labeling. In certain embodiments, fluorophore or chromophoredyes may be attached to one of the nucleotides added during the probehybridization, such that the probe hybridization to its target changesthe optical signature of beads (e.g., the fluorescent intensitieschange, thus providing changes in the optical signatures of the beads).

Described herein are methods and compositions to conduct accuratepolymorphism analysis for highly polymorphic target regions. Analogousconsiderations pertain to designs, compositions and methods ofmultiplexing PCR reactions.

The density of polymorphic sites in highly polymorphic loci makes itlikely that designated probes directed to selected polymorphic sites,when annealing to the target subsequence proximal to the designatedpolymorphic site, will overlap adjacent polymorphic sites. That is, anoligonucleotide probe, designed to interrogate the configuration of thetarget at one of the selected polymorphic sites, and constructed withsufficient length to ensure specificity and thermal stability inannealing to the correct target subsequence, will align with othernearby polymorphic sites. These interfering polymorphic sites mayinclude the non-designated selected sites as well as non-selected sitesin the target sequence.

The design of covering probe sets is described herein in connection withhybridization-mediated multiplexed analysis of polymorphisms in thescoring of multiple uncorrelated designated polymorphisms, as in thecase of mutation analysis for CF carrier screening. In this instance,the covering set for the entire multiplicity of mutations containsmultiple subsets, each subset being associated with one designated site.In the second instance, the covering set contains subsets constructed tominimize the number of probes in the set, as elaborated herein.

Arrays of bead-associated probes can be used in thehybridization-mediated analysis of a set of mutations within the contextof a large set of non-designated mutations and polymorphisms in theCystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Each ofthe designated mutations in the set is associated with the disease andmust be independently scored. In the case of a point mutation, twoencoded probes are provided to ensure alignment with the designatedsite, one probe complementary to the wild-type, the other to the mutatedor polymorphic target sequence.

In certain embodiments, the identification of the specific targetconfiguration encountered in the non-designated sites is of no interestso long as one of the sequences provided in the covering probe setmatches the target sequence sufficiently closely—and thus matches thetarget sequence exactly to ensure hybridization. In such a case, all orsome of the covering probes may be assigned the same code; in apreferred embodiment, such probes may be associated with the same solidsupport (“probe pooling”). Probe pooling reduces the number ofdistinguishable solid supports required to represent the requisitenumber of probes. In one particularly preferred embodiment, solidsupports are provided in the form of a set or array of distinguishablemicroparticles which may be decoded in situ. Inclusion of additionalprobes in the covering set to permit identification of additionalpolymorphisms in the target region is a useful method to elucidatehaplotypes for various populations.

Suitable probes may be designed to correspond to the known alleleswithin the CFTR gene locus. A number of polymorphisms and mutant allelesare known and available from literature and other sources.

Standard methods of temperature control are readily applied to set theoperating temperature of, or to apply a preprogramed sequence oftemperature changes to, single chips or to multichip carriers. Whencombined with the direct imaging of entire arrays of encoded beads asprovided in the READ™ format of multiplexed analysis, the application ofpreprogrammed temperature cycles provides real-time on-chipamplification of elongation products. Given genomic, mitochondrial orother DNA, linear on-chip amplification eliminates the need forpre-assay DNA amplification such as PCR, thereby dramatically shorteningthe time required to complete the entire typing assay. Time-sensitiveapplications such as cadaver typing are thereby enabled. Moreimportantly, this approach will eliminate the complexities of PCRmultiplexing, a limiting step in many genetic screening and polymorphismanalyses. In a preferred embodiment, a fluidic cartridge provides forsample and reagent injection, as well as temperature control.

The designs, compositions and methods described herein also pertain tothe multiplexed amplification of nucleic acid samples. In a preferredembodiment, covering sets of PCR primers composed of priming andannealing subsequences are used for target amplification.

Described below is a series of steps for selecting an appropriate arrayof probes and targets for hybridization analysis.

Task:

Identify (“select”) a set of probes, P, to perform one or moreconcurrent, “multiplexed” reactions permitting hybridization-mediatedinterrogation of nucleic acid sequences in order to determine thecomposition at each of a set of designated polymorphic sites,S={S₁,Y,S_(N)} said sites being located on M< or =N nucleic acid strandsT:={T₁, Y, T_(M)} (“targets”).

Targets—The collection, T, of targets, {T_(i)=(m_(i),σ_(i)); 1< or =i<or =M}, is generated in a polymerase chain reaction (PCR) using PCRprimers designed to place as many polymorphisms or mutations on eachsingle target under the condition that the target length l_(i) notexceed a preset maximal length, l_(max), and wherein the i-th target,T_(i) of length l_(i) is further characterized by:

-   -   a multiplicity, m_(i),        -   giving the number of said designated polymorphisms (or            mutations),        -   wherein Σ(i=1;i=M) m_(i)=N; and    -   an orientation, σ_(i), wherein        -   -   σ_(i)=+1 for a sense (“cis”) strand; or            -   σ_(i)=−1 for an anti-sense (“trans”) strand.

Probes—Mutation analysis preferably will involve the interrogation ofeach designated mutation site, S_(k), by hybridization of thecorresponding target to at least two designated interrogation probes,P_(k) ^(N) and P_(k) ^(V), of which at least a first probe, P_(k) ^(N),has a sequence that is complementary to the normal (“wild-type”)composition, and of which at least a second probe, P_(k) ^(V), has asequence that is complementary to a variant (“mutant”) composition. Inthe presence of polymorphisms or mutations at sites within theinterrogated subsequence other than the designated sites, it generallywill be desirable to provide “degenerate” probes matching theanticipated compositions at non-designated sites. The designationP_(k)=P_(k) (S_(k)) hereinafter is understood to refer to all probesdirected to the k-th designated site such that P_(k) is characterized bya number of probes, each of these probes having an orientation, σ_(ik),opposite to that of the cognate target.

Specifically, probes are to be selected, and probe-target reactions areto be configured in a manner involving one or more sets of reactions,each of these reactions being performed in a separate container, in sucha way as to minimize the interaction of any target subsequencecontaining a designated polymorphic site or mutation, S_(k), with anybut its corresponding designated probes, P_(k).

Strategy—While not necessarily generating an optimal configuration, thefollowing “heuristic” strategy provides the basis for a systematicprocess of assay optimization as a function of critical parametersincluding a maximal acceptable degree of similarity between twosequences, expressed in terms of a homology score, as well as a maximalacceptable level of “cross-hybridization”, manifesting itself inmagnitude of “off-diagonal” elements, P_(i) T_(j) of a co-affinitymatrix (see U.S. application Ser. No. 10/204,799, entitled:“Multianalyte molecular analysis using application-specific randomparticle arrays”) showing the degree of interaction between all probesand all targets in a given group or set.

To minimize cross-hybridization between any given target and probesdirected to other targets, distribute targets and their correspondingprobes—into a number, C, of containers in order to perform C separate“multiplexed” hybridization reactions, said number being chosen to be assmall as possible given a preset maximal acceptable level of sequencesimilarity (“maximal homology score”) between targets in the samecontainer.

To minimize cross-hybridization between any given target and probesdirected to other targets in the same container, switch the orientationof such other targets and that of their corresponding probes, allowingfor the possible reassignment of any target to another, possibly newcontainer.

Certain targets may have more than one region, each having a designatedprobe in the array which hybridizes with it. To minimizecross-hybridization as well as competitive hybridization within the samecontainer in such case, reduce the multiplicity of such an “offending”target by redesigning the PCR primer sets in order to produce two (ormore) smaller targets to replace the original single target, each of thenew targets having a lower multiplicity of hybridization regions thanthe original.

Implementation—The pseudocode below provides a description of theheuristic process of configuring the reaction so as to minimizecross-hybridization.

I - Assign targets - and cognate probes - to c sets (“Containers”) c =0; DO { REFSEQ = SelectTarget(T); /* randomly pick a target sequencefrom given collection, T */ ShrinkCollection (T, 1); /* remove selectedtarget from collection */ S = L(c); InitializeList (L(g), REFSEQ); /*place selected target into new set (“group”)implemented in the form of alist, S */ AlignTargets (REFSEQ, T, HScores); /* align remaining targetsto REFSEQ by pairwise alignment or multiple sequence alignment; returnhomology scores */ SortTargets (HScores, T); /* rank target seq's in theorder of increasing homology score with respect to REFSEQ; first entryleast similar, last entry most similar to REFSEQ */ t = AssignTargets(maxHSCORE, S, , T); /* remove targets from collection in the order ofincreasing homology scores up to maxHScore and place them into the list,starting at top; return number of targets assigned to the list, S */ShrinkCollection (T , t); /* remove t selected targets from collection*/ c++; } WHILE ( T not EMPTY);Optionally, one or more lists may be pruned should they contain morethan an acceptable number of targets (for example, if it is determined,based on too many targets in a list, that maxHScore should be lowered)by removing targets from the bottom of one or more of the lists andplacing them back into the collection T.

II - Refine group configuration FOR ( i=0; i<c; i++) /*examine eachgroup in turn */ { S = L(i); /* List S holds targets in current group */PofS = SelectProbes ( P, S); /* select from P all probes directed totargets in current list */ /* each probe is designed to match at leastone target - this is referred to as the probe cognate for that target;NOTE: refers to diagonal elements in co-affinity matrix */ WHILE (S notEMPTY) { T = PopTarget (S); PerformProbeTargetRxn (PofS, T) /* place Tin contact with all selected probes, preferably arranged in a probearray */ FOR (each probe, P, in PofS) { I = DetermineInteractionStrength(P, T); /* eliminate unacceptably large off-diagonal element inco-affinity matrix*/ IF( (P not cognate to T) AND (I > maxI) ) {FlipOrientation (P); /* flip probe orientation */ FlipOrientation (TcP);/* flip orientation of target cognate to P */ } } /* check “flipped”targets in list S*/ flippedT = PopTarget (S); PerformProbeTargetRxn(PofS, flippedT) /* place T in contact with all selected probes,preferably arranged in a probe array */ FOR (each probe, P, in PofS) { I= DetermineInteractionStrength (P, flippedT); /* eliminate unacceptablylarge off- diagonal element in co- affinity matrix*/ IF( (P not cognateto flippedT) AND (I > maxI) ) { PushTarget (flippedT, TempList); /*Place flipped target into temporary list */ } } } } S = TempList; /*List S holds flipped targets in temp list */ /* Check targets in templist */ WHILE (S not EMPTY) { T = PopTarget (S); FOR (j=0; j <c; j++) {IF( L(j) != L(T) ) /* Check T against probes in all existing lists withexception of those in T's original list */ { L = L(j); PofL =SelectProbesFrom List (L); /* select probes in Group L */PerformProbeTargetRxn (PofL, T) /* place T in contact with all selectedprobes, preferably arranged in a probe array */ FOR (each probe, P, inPofL) { I = DetermineInteractionStrength (P, T); /* eliminateunacceptably large off-diagonal element in co-affinity matrix*/ IF( (Pnot cognate to T) AND (I> maxI) ) { FlipOrientation (T)); /* flip targetorientation back to original */ FlipOrientation (PcT); /* fliporientation of p r o b e cognate to target T */ PushTarget(T, NewList);/* init new group */ } } } } } FlipOrientation (P); /* flip probeorientation */ FlipOrientation (TcP); /* flip orientation of targetcognate to P */

EXAMPLE I CFTR Assay

Genomic DNA extracted from several patients was amplified withcorresponding primers in a multiplex PCR (mPCR) reaction. The PCRconditions and reagent compositions were as follows:

PRIMER DESIGN: One of the primers (sense or antisense, depending ondesign considerations, discussed below) was modified with a label (suchas, Cy3, Cy5 and Cy5.5) at the 5′ end and the corresponding primer forthe complementary sequence had a phosphate group added at the 5′ end, sothat the amplicon could be digested by λ exonuclease during post-PCRprocessing of the target (see below). Hybridization was detected bydetection of the dyes (Cy3, Cy5 or Cy5.5) in the hybridized product.Multiplex PCR (mPCR) was performed in two groups with the followingprimers (Tables I and II), and with the reagents and under theconditions listed below. The exon number where the mutation is locatedappears below in the left-had column of Tables I and II.

TABLE I Artificial sequence artificial primer EX-5-1-CyGTC AAG CCG TGT TCT A GAT SEQ ID NO.: 1 EX-5-2-PGTT GTA TAA TTT ATA ACA ATA GT SEQ ID NO.: 2 EX-7-1-PAC TTC AAT AGC TCA GCC TTC SEQ ID NO.: 3 EX-7-2-CyTAT GGT ACA TTA CCT GTA TTT TG SEQ ID NO.: 4 EX-9-1-PTGG TGA CAG CCT CTT CTT SEQ ID NO.: 5 EX-9-2-CyGAA CTA CCT TGC CTG CTC CA SEQ ID NO.: 6 EX-12-1-PTCT CCT TTT GGA TAC CTA GAT SEQ ID NO.: 7 EX-12-2-CyTGA GCA TTA TAA GTA AGG TAT SEQ ID NO.: 8 EX-13-1-PAGG TAG CAG CTA TTT TTA TGG SEQ ID NO.: 9 EX-13-2-CyATC TGG TAC TAA GGA CAG SEQ ID NO.: 10 EX-14B-1-PTCT TTG GTT GTG CTG TGG CT SEQ ID NO.: 11 EX-14B-2-CyACA ATA CAT ACA AAC ATA GT SEQ ID NO.: 12 EX16A-1PCTT CTG CTT ACC ATA TTT GAC SEQ ID NO.: 13 EX16A-2-CyTAAT ACA GAC ATA CTT AAC G SEQ ID NO.: 14 EX-18-1-PGG AGA AGG AGA AGG AAG AG T SEQ ID NO.: 15 EX18-2-CyATC TAT GAG AAG GAA AGA AGA SEQ ID NO.: 16 Ex-19-1-CyGGC CAA ATG ACT GTC AAA GA SEQ ID NO.: 17 Ex-19-2-PTGC TTC AGG CTA CTG GGA TT SEQ ID NO.: 18 mPCR Group I Primers: (“Cy”denotes a dye label, and “P” denotes a phosphate modification, at the 5′end of the primer)

TABLE II  mPCR Group II Primers: Ex-3-1-Cy C GGC GAT GTT TTT TCT GGA GSEQ ID NO.: 19 Ex-3-2-P T ACA AAT GAG ATC CTT ACC C SEQ ID NO.: 20Ex-4-1-P AGC TTC CTA TGA CCC GGA TA SEQ ID NO.: 21 Ex-4-2-CyTGT GAT GAA GGC CAA AAA TG SEQ ID NO.: 22 EX-10-1-PTGT TCT CAG TTT TCC TGG AT SEQ ID NO.: 23 EX-10-2-Cy CTC TTC TAG TTG GCA TGC TT SEQ ID NO.: 24 Ex-11-1-PCAG ATT GAG CAT ACT AAA AG SEQ ID NO.: 25 EX11-2-CyAC ATG AAT GAC ATT TAC AGC SEQ ID NO.: 26 Int-19-1-CyAA TCA TTC AGT GGG TAT AAG C SEQ ID NO.: 27 Int-19-2-PCCT CCT CCC TGA GAA TGT TGG SEQ ID NO.: 28 EX-20-1-PC TGG ATC AGG GAA GA GAA GG SEQ ID NO.: 29 EX20-2-CyTCC TTT TGC TCA CCT GTG GT SEQ ID NO.: 30 EX21-1-PTGA TGG TAA GTA CAT GGG TG SEQ ID NO.: 31 EX21-2-CyCAA AAG TAC CTG TTG CTC CA SEQ ID NO.: 32

PCR Master Mix Composition

For 20 μl reaction/sample:

Components Volume (μl) 10X PCR buffer 2.0 25 mM MgCl₂ 1.4 dNTPs (2.5 mM)4.0 Primer mix (Multiplex 10x) 3.0 Taq DNA polymerase 0.6 ddH2O 3.0 DNA6.0 Total 20

PCR Cycling Conditions

Hot Start 94° C. 15 min 94° C. 30 sec, 60% ramp 60° C. 30 sec, 50% ramp{close oversize bracket} 30 cycles 72° C. 50 sec, 35% ramp 72° C.  8 min

Amplifications were performed using a Perkin Elmer 9700 thermal cycler.Optimal primer concentrations were determined for each primer pair. Thereaction volume can be adjusted according to experimental need.

-   Post PCR processing: Following amplification, PCR products were    purified using either a QIAquick PCR purification kit (QIAGEN, Cat    #28104), or by Exonuclease 1 treatment (Amersham). For the latter    procedure: an aliquot of 8 μl of PCR product was added in a clean    tube with 2.5 μl of Exonuclease 1 (Amersham), incubated at 37° C.    for 15 minutes and denatured at 80° C. for 15 min. Thereafter,    single stranded DNA was generated as follows:

PCR reaction products were incubated with 2.5 units of λ exonuclease in1× buffer at 37° C. for 20 min, followed by enzyme inactivation byheating to 75° C. for 10 min. Under these conditions, the enzyme digestsone strand of duplex DNA from the 5′-phosphorylated end and releases5′-phosphomononucleotides (J. W. Little, et al., 1967). Single-strandedtargets also can be produced by other methods known in the art, althoughheating the PCR products to generate single stranded DNA, isundesirable. The single stranded DNA can be used directly in the assay.

-   ON CHIP Hybridization—The CFTR gene sequence from Genebank    (www.ncbi.nlm.nih.gov) was used to model the wild-type. The 52    probes were divided into two groups on the basis of their sequence    homologies, in accordance with the “heuristic” probe selection    algorithm, i.e., in such a way as to avoid overlapping homologies    among different probes to the extent possible. The mutations    included in each group were selected so as to minimize overlap    between probe sequences in any group and thereby to minimize    intra-group cross-hybridization under multiplex assay conditions.

Probe sequences were designed by PRIMER 3.0 software (seehttp://www.genome.wi.mit.edu incorporated herein by reference), seekingto include the following characteristics in each probe:

-   (b) a mismatch in the center of the probe;-   (c) probe length 16-21 bases;-   (d) low self compatibility;-   (e) 30-60% GC content; and-   (f) no more than three consecutive identical bases.-   Each probe sequence was aligned with its complementary exon    sequence. See    http://mbcr.bcm.tmc.edu;http://searchlauncher.bcm.tmc.edu/seq-search/alignment.html,    incorporated herein by reference. The percent homology between each    probe and non-desired target sequences (i.e., those sequences    representing mutations other than those which the probe is intended    to hybridize with) was calculated, and probes were selected such    that the percent homology between probes for each mutation and    non-desired target sequences on the same array was less than 50%.

Probe selection was further refined based on the heuristic selectionalgorithm, set forth above. Probe selection was also refined in part onexperimental selection, and in part on consideration that certainmismatched base pairs, particularly, G-T, will tend to be stable. Ininstances where probes could hybridize incorrectly with mismatchesforming a G-T pairing, and in certain other instances, the anti-senseprobes were used, rather than the sense probes, if such stablemismatches could be avoided, or if it was experimentally demonstratedthat incorrect hybridization was eliminated by using the antisenseprobe. The cases where antisense probes were used are indicated in theProbe Sequence Table III below.

Wild type and mutant probes for 26 CF mutations were synthesized witheither 5′ Biotin-TEG or amine modification at the 5′ end (Integrated DNATechnologies). Different bead chemistry can use a different 5′ end, suchthat a biotin modification is coupled to beads coated with neutravidin,and an amine modification is coupled to beads coated with BSA. Probeswere dissolved in 1× TE or dsH₂O at a concentration of 100 μM. Analiquot of 100 μl of 1% bead solids, for each type of bead, was washedthree times with 500 μl of TBS-1 (1× TE, 0.5 M NaCl2). Probes were addedto 500 μl bead suspension and incubated at room temperature for 45-60minutes on a roller. Beads were washed once with wash solution TBS-T (1×TE, 0.15 M NaCl₂, 0.05% Tween 20) or PBS-T (Phosphate buffered saline,Tween 20) and twice with TBS-2 (1× TE, 0.15 M NaCl₂) and re-suspended in1× TBS-2. Beads were assembled on the surface of chips as describedearlier. The probes were also divided into two groups and assembled ontwo separate chips. A third group was assembled for reflex testincluding 5T/7T/9T polymorphisms. Negative and positive controls werealso included on the chip surface, and assay signal was normalized usingthese controls. For negative controls, beads were coupled with a 10-merstrand of dCTP (Oligo-C) and immobilized on the chip surface. For apositive control signal, the human β Actin sequence was used. The signalfrom Oligo-C was used as the background to subtract the noise level andβ Actin was used to normalize the data.

TABLE III A - Hybridization Group I: Bead cluster Mutation 1OLIGO-C(control) 2 BA 3 OligoC-1 4 G85E-WT 5 G85E-M 6 621 + 1G > T-WT 7621 + 1G > T-M 8 R117H-WT 9 R117H-M 10 I148-WT 11 I148-M 12 A455E-WT 13A455E-M 14 508-WT 15 OLIGOC-2 16 F508 17 I507 18 G542-WT 19 G542-M 20G551D-WT 21 G551D-M 22 R560-WT 23 R560-M 24 R553X-WT 25 R553X-M 26OLIGOC-3 27 1717 − 1G > A-WT 28 1717 − 1G > A-M 29 3849 + 10kb-WT 303849 + 10kb-M 31 W1282X-WT 32 OLIGOC-4 33 W1282X-M 34 N1303K-WT 35N1303K-M 36 OLIGOC-5 B - Hybridization Group II Bead Cluster Mutation 1BA 2 1898 + 5G -WT 3 OLIGO-C(control) 4 1898 + 5G-M 5 OLIGO-C-1 6R334W-WT 7 R334W-M 8 1898 + 1G > A-WT 9 1898 + 1G > A-WT 10 1078delT-M11 OLIGO-C-2 12 D1152-WT 13 D1152-M 14 R347P-WT 15 R347P-M 16 711 + 1G >T-WT 17 711 + 1G > T-M 18 3659delC-WT 19 3659delC-M 20 OLIGO-C-3 21R1162X-WT 22 R1162X-M 23 2789 + 5G-WT 24 2789 + 5G-M 25 3120 + 1G > A-WT26 3120 + 1G > A-WT 27 OLIGO-C-4 28 A455E-WT 29 A455E-M 30 2184delA-WT31 2184delA-M 32 1078delT-WT 33 OLIGO-C-5 C - Hybridization Group III(total 6 groups) Cluster # Mutation i β Actin 1 Oligo C 2 5T 3 7T 4 9TProbe sequences for detecting each mutation were as follows (probes tosense or antisense sequences were selected as described above):

TABLE IV NORMAL/VARIANT SEQUENCE CAPTURE PROBES EX-3AT GTT CTA TGG AAT CTT TT TA SEQ ID NO.: 33 G85EAT GTT CTA TGA AAT CTT TT TA SEQ ID NO.: 34 EX-4TA TAA GAA GGT AAT ACT TC CT SEQ ID NO.: 35 621-MTA TAA GAA GTT AAT ACT TC CT SEQ ID NO.: 36 INT-4CC TCA TCA CAT TGG AAT GC AG SEQ ID NO.: 37 I148TCC TCA TCA CAC TGG AAT GC AG SEQ ID NO.: 38 EX-4CAA GGA GGA ACG CTC TAT CG C SEQ ID NO.: 39 R117HCAA GGA GGA ACA CTC TAT CG C SEQ ID NO.: 40 EX-5ATG GGT ACA TAC TTC ATC AA A SEQ ID NO.: 41 711+1GATG GGT ACA TAA TTC ATC AA A SEQ ID NO.: 42 EX-7GAA TAT TTT CCG GAG GAT GAT SEQ ID NO.: 43 334-MGAA TAT TTT CCA GAG GAT GAT SEQ ID NO.: 44 EX-7CAT TGT TCT GCG CAT GGC GGT SEQ ID NO.: 45 347-MCAT TGT TCT GCC CAT GGC GGT SEQ ID NO.: 46 EX-7CT CAG GGT TCT TTG TGG TG TT SEQ ID NO.: 47 1078DEL TCT CAG GGT TC TTG TGG TG TT SEQ ID NO.: 48 EX-9ACA GTT GTT GGC GGT TGC TGG SEQ ID NO.: 49 A455EACA GTT GTT GGA GGT TGC TGG SEQ ID NO.: 50 EX-10AAA GAA AAT ATC ATC TTT GGT SEQ ID NO.: 51 F508AAA GAA AAT ATC ATT GGT GT SEQ ID NO.: 52 1507AAA GAA AAT ATC TTT GGT GT SEQ ID NO.: 53 EX-12ATA TTT GAA AGG TAT GTT CT TT SEQ ID NO.: 54 1898+1ATA TTT GAA AGA TAT GTT CT TT SEQ ID NO.: 55 Ex-13GAA ACA AAA AAA CAA TCT TTT SEQ ID NO.: 56 2184 delAGAA ACA AAA AA CAA TCT TTT SEQ ID NO.: 57 EX-14BTTG GAA AGT GAG TAT TCC ATG SEQ ID NO.: 58 2789+5GTTG GAA AGT GAA TAT TCC ATG SEQ ID.NO.: 59 EX-16ACT TCA TCC AGA TAT GTA AAA SEQ ID NO.: 60 31120+1G/AACT TCA TCC AGG TAT GTA AAA SEQ ID NO.: 61 Ex-11TAT AGT TCT TGG AGA AGG TGG SEQ ID NO.: 62 G542XTAT AGT TCT TTG AGA AGG TGG SEQ ID NO.: 63 EX-11TCT TTA GCA AGG TGA ATA ACT SEQ ID NO.: 64 R560TCT TTA GCA ACG TGA ATA ACT SEQ ID NO.: 65 EX-11-553/551GAG TGG AGG TCA ACG AGC AAG SEQ ID NO.: 66 G551DGAG TGG AGA TCA ACG AGC AAG SEQ ID NO.: 67 R553XGTG GAG GTC AAT GAG CAA GA SEQ ID NO.: 68 EX-11TGG TAA TAG GAC ATC TCC AAG SEQ ID NO.: 69 1717-MTGG TAA TAA GAC ATC TCC AAG SEQ ID NO.: 70 EX-18ACT CCA GCA TAG ATG TGG ATA SEQ ID NO.: 71 1152XACT CCA GCA TAC ATG TGG ATA SEQ ID NO.: 72 EX-19-SENSEGAA CTG TGA GCC GAG TCT TTA SEQ ID NO.: 73 R1162XGAA CTG TGA GCT GAG TCT TTA SEQ ID NO.: 74 EX-19TGG TTG ACT TGG TAG GTT TAC SEQ ID NO.: 75 3659TGG TTG ACT TG TAG GTT TAC SEQ ID NO.: 76 INT-19T TAA AAT GGT GAG TAA GA CAC SEQ ID NO.: 77 3849T TAA AAT GGC GAG TAA GA CAC SEQ ID NO.: 78 EX-20TGC AAC AGT GGA GGA AAG CCT SEQ ID NO.: 79 1282XTGC AAC AGT GAA GGA AAG CCT SEQ ID NO.: 80 EX-21A TTT AGA AAA AAC TTG GAT CC SEQ ID NO.: 81 N1303KA TTT AGA AAA AAG TTG GAT CC SEQ ID NO.: 82 β A-PROBEAG GAC TCC ATG CCC AG SEQ ID NO.: 83

The hybridization buffer has been optimized for use in uniplex and/ormultiplex hybridization assays and is composed of (finalconcentrations): 1.125 M Tetramethyl-Ammonium Chloride (TMAC), 18.75 mMTris-HCL (pH 8.0), 0.75 mM EDTA (pH 8.0) and 0.0375% SDS. Ten μl ofhybridization mixture containing buffer and ssDNA was added on the chipsurface and incubated at 55° C. for 15 minutes. This is a shorterhybridization time than the several hours normally used, because longerhybridization times tend to generate uncontrolled excess hybridization.The chip was washed with 1× TMAC buffer three times, covered with aclean cover slip and analyzed using a BAS imaging system. Images areanalyzed to determine the identity of each of the probes. The resultsare shown below in FIGS. 1 and 2.

Each allele of a given mutation was analyzed as follows. First, thesignal from the hybridized alleles was corrected as follows:

-   -   (i) Signal for allele A (labeled amplicon)=Raw signal from        labeled amplicon-hybrid minus raw counts from negative        (background) control    -   (ii) Signal for allele B (unlabeled amplicon)=Raw signal from        unlabeled amplicon-hybrid minus raw counts from negative        (background) control        Then an allelic ratio was calculated:

Allelic ratio=Signal for allele A/Signal for allele B

When the value of (i) was less than or equal to zero, it was adjusted to0.01 to avoid the generation of negative values. Allelic ratios of >2were scored as homozygous for allele A (indicating mutant/polymorph),while an allelic ratio of <0.5 was scored as homozygous for allele B(wild type). An allelic ratio of 0.8 to 1.2 was scored as heterozygous.Values which fell in between these thresholds were considered ambiguousand the assay was repeated.

EXAMPLE II Screening of Multiple Patient Samples—Side-by-Side Comparisonof hMAP with Dot Blot Analysis

A number of patient samples were obtained and amplified for simultaneousscreening. The method of amplification and primer design was asdescribed above. After amplification, analysis techniques on sampleswere compared for 26 CFTR mutations. A set was analyzed usingconventional dot blot hybridization methods, and the same set wasanalyzed with the methods and reagents of the invention. The results foreach patient sample were compiled and both results were compared. Therewas 100% concordance with the two methods of detection. The number ofsamples identified as positives for each mutation are listed in Table V.

TABLE V Comparison of Testing of Samples Samples tested by dot-blot andmethods described herein Mutations # Positives # Negatives Total G85E 1111 22 G85E/621 + 1G 8 8 621 + 1G > T 11 13 24 621 + 1G > T/delF508 2 2R117H 19 19 R117H/delF508 1 1 I148T 48 48 delF508 58 14 72 I507 11 11delF508/R560 1 1 G542X 44 11 55 G551D 11 11 R553X 15 15 1717 − 1G > A 1414 R560T 9 9 3849 + 10kbC > T 25 14 39 W1282X 53 13 66 N1303K 31 15 46mPCR-WT 87 87 711 + 1G > T 19 9 28 711 + 1G > T/621 + 1G 1 1 R334W 19 1130 R347P 13 13 1078delT 11 11 A455E 18 11 29 1898 + 1G > A 24 10 342184delA 10 10 20 2789 + 5G > A 20 10 30 3120 + 1g > A 18 10 28 R1162X13 8 21 3569delC 8 8 D1152 47 9 56 mPCR-WT 80 80 TOTAL 939

It should be understood that the terms, expressions and examplesdescribed herein are exemplary only and not limiting and that processesand methods can be performed in any order, unless the sequence of stepsis specified. The invention is defined only in the claims which followand includes all equivalents of the claims.

1. A method of optimizing hybridization analysis for detecting knowngenetic mutations and polymorphisms, wherein the following steps areinitially performed: a) providing a set of oligonucleotide primer pairs,each pair capable of annealing with complementary polynucleotide strandsto delineate a region of the corresponding target which includes atleast one designated mutation or polymorphic site; b) contacting saidset of oligonucleotide primer pairs with said targets under conditionsallowing formation of amplicon pairs, each amplicon pair comprising adesignated amplicon sense strand corresponding to either a target senseor antisense strand and an amplicon antisense strand corresponding tothe other target strand (either a sense or antisense target stand); c)selecting two groups of encoded probes wherein probes having differentcodes have different nucleotide sequences, sense probes selected suchthat each sense probe is complementary, in whole or in substantial part,to an amplicon antisense strand or a subsequence thereof (referred to asa “complementary amplicon antisense strand” and other antisenseamplicons referred to as “non-designated amplicons”), and antisenseprobes selected such that each antisense probe is complementary, inwhole or in substantial part, to an amplicon sense strand or asubsequence thereof (referred to as a “complementary amplicon sensestrand” and other sense amplicons referred to as “non-designatedamplicons”); and wherein the method comprises: reducingcross-hybridization between probes and non-designated amplicons bydistributing sense probes and complementary amplicon antisense strandsinto more than one different containers so as to perform separatehybridization reactions in different containers, said number ofcontainers being as small as possible while providing that the sequencesimilarity between amplicons (and probes) in the same container notexceed a preset acceptance level.
 2. The method of claim 1 wherein thepolynucleotide is an mRNA, a cDNA or a double-stranded polynucleotide,including DNA.
 3. The method of claim 1 wherein probes having differentsequences are encoded by associating probes to carriers, includingbeads, said carriers having different optical signatures.
 4. The methodof claim 3 wherein the encoding is with color.
 5. The method of claim 1wherein one primer in a primer pair is labeled at the 5′ end with alabel and the other primer in the primer pair has a phosphatemodification at the 5′ end.
 6. The method of claim 5 wherein theamplicon incorporating said phosphate modified primer is digested. 7.The method of claim 1 or 2 wherein the hybridization of amplicons andprobes is determined by detecting signals from the labels associatedwith amplicons.
 8. A method of optimizing hybridization analysis fordetecting known genetic mutations and polymorphisms, wherein thefollowing steps are initially performed: a) providing a set ofoligonucleotide primer pairs, each pair capable of annealing withcomplementary polynucleotide strands to delineate a region of thecorresponding target which includes at least one designated mutation orpolymorphic site; b) contacting said set of oligonucleotide primer pairswith said targets under conditions allowing formation of amplicon pairs,each amplicon pair comprising a designated amplicon sense strandcorresponding to either a target sense or antisense strand and anamplicon antisense strand corresponding to the other target strand(either a sense or antisense target stand); c) selecting two groups ofencoded probes wherein probes having different codes have differentnucleotide sequences, sense probes selected such that each sense probeis complementary, in whole or in substantial part, to an ampliconantisense strand or a subsequence thereof (referred to as a“complementary amplicon antisense strand” and other antisense ampliconsreferred to as “non-designated amplicons”), and antisense probesselected such that each antisense probe is complementary, in whole or insubstantial part, to an amplicon sense strand or a subsequence thereof(referred to as a “complementary amplicon sense strand” and other senseamplicons referred to as “non-designated amplicons”); and wherein themethod comprises: maintaining the degree of sequence similarity andcross-hybridization between probes and non-designated amplicons below apreset acceptance level, by substituting one or more antisense probesfor sense probes (and substituting the complementary amplicon sensestrands for amplicon antisense strands).
 9. The method of claim 8wherein the substituted antisense probes are complementary, in whole orin substantial part, to the substituted sense probes.
 10. The method ofclaim 8 wherein the polynucleotide is an mRNA, a cDNA or adouble-stranded polynucleotide, including DNA.
 11. The method of claim 8wherein probes having different sequences are encoded by associatingprobes to carriers, including beads, said carriers having differentoptical signatures.
 12. The method of claim 11 wherein the encoding iswith color.
 13. The method of claim 8 wherein one primer in a primerpair is labeled at the 5′ end with a label and the other primer in theprimer pair has a phosphate modification at the 5′ end.
 14. The methodof claim 13 wherein the amplicon incorporating said phosphate modifiedprimer is digested.
 15. The method of claim 8 wherein the hybridizationof amplicons and probes is determined by detecting signals from thelabels associated with amplicons.
 16. A method of optimizinghybridization analysis for detecting known genetic mutations andpolymorphisms, comprising the following: a) providing a set ofoligonucleotide primer pairs, each pair capable of annealing withcomplementary polynucleotide strands to delineate a region of thecorresponding target which includes at least one designated mutation orpolymorphic site; b) contacting said set of oligonucleotide primer pairswith said targets under conditions allowing formation of amplicon pairs,each amplicon pair comprising a designated amplicon sense strandcorresponding to either a target sense or antisense strand and anamplicon antisense strand corresponding to the other target strand(either a sense or antisense target stand), and wherein, in order tomaintain the degree of sequence similarity and cross-hybridization withpartially complementary non-designated probes to below a presetacceptance level, the number of regions in an amplicon designated forhybridization with probes is controlled.
 17. The method of claim 16wherein the number of regions in an amplicon designated forhybridization with probes is determined, and then amplicons with fewersuch regions are generated in a subsequent step.
 18. A method ofdesigning a probe array for use in hybridization analysis for detectinggenetic mutations and polymorphisms, wherein the following steps areinitially performed: a) providing a set of oligonucleotide primer pairs,each pair capable of annealing with complementary polynucleotide strandsto delineate a region of the corresponding target which includes atleast one designated mutation or polymorphic site; b) contacting saidset of oligonucleotide primer pairs with said targets under conditionsallowing formation of amplicon pairs, each amplicon pair comprising adesignated amplicon sense strand corresponding to either a target senseor antisense strand and an amplicon antisense strand corresponding tothe target strand (either a sense or antisense target stand); c)selecting two groups of encoded probes wherein probes having differentcodes have different nucleotide sequences, sense probes selected suchthat each sense probe is complementary, in whole or in substantial part,to an amplicon antisense strand or a subsequence thereof (referred to asa “complementary amplicon antisense strand” and other antisenseamplicons referred to as “non-designated amplicons”), and antisenseprobes selected such that each antisense probe is complementary, inwhole or in substantial part, to an amplicon sense strand or asubsequence thereof (referred to as a “complementary amplicon sensestrand” and other sense amplicons referred to as “non-designatedamplicons”); and wherein the method comprises the following steps: a)examining the degree of homology between individual sense probes, orbetween individual amplicon sense strands; b) dividing members of thesense probes into one or more probe sets, and dividing the complementaryamplicon sense strands into corresponding sets, said division performedso as to maintain the degree of sequence similarity between members ofeach probe set (and between members of each amplicon strand set) below apreset acceptance level; c) performing the following steps: A(i)determining whether, upon contacting under hybridizing conditions, amember of an amplicon set with the corresponding probe set; or, whether,upon contacting under hybridizing conditions, a member of a probe setwith the corresponding amplicon set, the degree of cross-hybridizationof said member with non-designated members of said probe set or saidamplicon set, as applicable, will exceed a preset acceptance level; and,if not: A(ii) retaining, in the respective sets, said members of saidamplicon set and said members of said probe set, and repeating step(A)(i) for another member of said amplicon set or for another member ofsaid probe set; (B)(i) but if said degree of cross-hybridization doesexceed said acceptance level: replacing, in said respective sets, thecross-hybridizing probe with a complementary antisense probe, orreplacing the cross-hybridizing antisense amplicon strand with acomplementary amplicon sense strand; and (B)(ii) repeating step (A)(i)with the replacement probes and amplicons, and if the degree ofcross-hybridization does not exceed the acceptance level: retaining saidantisense probe and said designated member anti-sense amplicon in theirrespective sets and repeating step (A)(i) for another member of saidamplicon set; (B)(iii) but if the degree of cross-hybridization exceedsthe acceptance level after repeating step (A)(i) with said replacementprobes and amplicons: determining whether, upon contacting saidreplacement probes and amplicons, respectively, with probes in any otherset of probes, or amplicons in any other set of amplicons, asapplicable, the degree of cross-hybridization does not exceed theacceptance level, and if so, retaining said replacement members in theirrespective sets; but if the degree of cross-hybridization exceeds theacceptance level following such determination, placing the replacementmembers into a new set, and (C) repeating steps (A)(i) to (B)(iii), foranother member of said amplicon set or said probe set, as applicable.19. The method of claim 18 further including the steps of: providingconditions capable of generating two subgroups (respectively, designated“WT” and “MP”) of each of the set of amplicon sense strands and the setof amplicon antisense strands, where the subgroups differ in sequence atone or more positions, where amplicons in a WT subgroup correspond witha wild-type region in the genomic sequence and where amplicons in a MPsubgroup correspond with a mutant or polymorphic region in the genomicsequence; selecting four subgroups of probes (designated, respectively,WT sense, WT antisense, MP sense, MP antisense) such that probes in eachsubgroup are complementary, in whole or in substantial part, to acorrespondingly labeled but complementary subgroup of amplicon strands,or to a subsequence thereof; determining: (i) whether the level ofcross-hybridization between a WT amplicon antisense strand and thesubstantially complementary MP sense probe, or between an MP antisenseamplicon and the substantially complementary WT sense probe, will exceedan acceptance level and, if so, (ii) determining whether said level ofcross-hybridization will fall within the acceptance level if said MPsense or said WT sense probes are replaced with, respectively, acomplementary WT antisense probe or a complementary MP antisense probe;and if so, (iii) determining whether said WT antisense probe or said MPantisense probe, as applicable, will, respectively, exceed theacceptance level for cross-hybridization with other MP sense ampliconsor other WT sense amplicons, and if so, (iv) determining whether placingsaid WT antisense probe or said MP antisense probe into a separatesubgroup together with complementary MP amplicons or WT amplicons, asapplicable, together with any other probes and amplicons selected bysteps (i) to (iv) for said separate subgroup, will exceed the acceptancelevel for cross-hybridization with said other probes and amplicons insaid separate subgroup, and if not: proceeding with said separation intosaid separate subgroup; but if so, (v) repeating step (iv) using anotherseparate subgroup, and proceeding with said separation into said anotherseparate subgroup for probes and amplicons until the acceptance level ismet.
 20. The method of claim 18 or 19 wherein the determination of theacceptance level includes reducing or minimizing the number of G-T basepairing.
 21. The method of claim 18 or 19 wherein the acceptance levelis determined using the computer program PAM™.
 22. The method of claim18 wherein the polynucleotide target is an mRNA, cDNA or adouble-stranded polynucleotide, including DNA.
 23. The method of claim18 wherein probes are encoded by associating probes with differentsequences to carriers, including beads, said carriers having differentoptical signatures.
 24. The method of claim 23 wherein the encoding iswith color.
 25. The method of claim 18 or 19 wherein one primer in aprimer pair is labeled at the 5′ end with a label and the other primerin the primer pair has a phosphate modification at the 5′ end.
 26. Themethod of claim 25 wherein the amplicon including the primer with thephosphate modification is digested.
 27. The method of claim 18 or 19wherein the hybridization of amplicons and probes is determined bydetecting signals from the labels associated with amplicons.
 28. Themethod of claim 25 wherein the labels are Cy3, Cy5 and Cy5.5.
 29. Themethod of claim 19 wherein the WT probes and the MP probes which areclosest in sequence differ at only one nucleotide position.
 30. Themethod of claim 19 wherein the WT amplicons and the MP amplicons whichare closest in sequence differ at only one nucleotide position.
 31. Aset of probes or amplicons selected by the process set forth in any ofclaims 1 to
 30. 32. Probes for screening samples for CFTR mutationsassociated with cystic fibrosis having sequences as set forth in SEQ IDNos. 33 to
 83. 33. A method of testing for mutations or polymorphisms ina locus using an array of carrier-displayed probe pairs, with differentcarriers displaying different members of a probe pair, wherein one probein a pair can be used to identify, by way of hybridization, a designatednormal allele and the other probe can be used to identify, by way ofhybridization, a counterpart designated variant allele, said carriersbeing encoded to identify the probes displayed thereof, comprising:amplifying the genomic regions corresponding to said designated allelesfrom a sample suspected to have mutations of interest, using two primersfor each said region, wherein one of the primers is labeled at its 5′end, to produce a set of labeled amplicons; producing single-strandedamplicons; placing the carrier-displayed probe pairs on a substrate;contacting, for a time which does not substantially exceed that neededto achieve hybridization between probes and amplicons, the bound arrayof probe pairs with the amplicons under hybridizing conditions;detecting hybridization of probes and amplicons based on signals fromthe labeled amplicons which hybridize to the probe array; and decodingthe array to determine the identities of the hybridized amplicons, andthereby to determine the corresponding mutations or polymorphisms. 34.The method of claim 33 wherein single stranded amplicons are produced bydigestion of one of the amplicon strands.
 35. The method of claim 34wherein an amplicon strand is preselected for digestion byphosphorylating the primer incorporated in it.
 36. The method of claim35 wherein the digestion is with λ Exonuclease.
 37. The method of claim33 wherein the locus is in the CFTR region.
 38. The method of claim 33wherein the carrier-displayed probe pairs are affixed to the substrate.39. The method of claim 33 wherein the reacting time does not exceed 15minutes.
 40. The method of claim 33 wherein the carriers are microbeads.41. The method of claim 33 wherein the substrate and bound carriers canbe viewed under a microscope.
 42. The method of claim 33 wherein saidgenomic regions are mRNA (or cDNA derived therefrom) or adouble-stranded polynucleotide, including DNA.
 43. The method of claim38 wherein the microbeads are encoded with different optical signatures.44. The method of claim 33 wherein the encoding is with color.
 45. Amethod of differentiating homozygous, heterozygous and wild-type (formutant or polymorphic or wild-type alleles in a target sample) usingresults obtained from a probe array designed to detect designated mutantor polymorphic alleles, and wild-type alleles, through hybridization ofprobes and targets, where such results are include compensation formismatched probe-target binding, comprising: amplifying the genomicregions in the target sample predicted to include either the designatedmutant or polymorphic alleles or the corresponding wild-type alleles, toproduce labeled amplicons corresponding to the designated mutant orpolymorphic alleles (“mutant/polymorphic amplicons”) and labeledamplicons corresponding to the wild-type alleles (“wild-typeamplicons”); providing an array of probe pairs, one member beingcomplementary to a mutant/polymorph amplicon and the other member beingcomplementary to the corresponding wild-type amplicon; contacting thearray probe pairs with the amplicons; detecting, for wild-type andmutant/polymorph amplicons, binding based on the presence of signalsfrom the labeled bound amplicons, said signal being corrected to adjustfor mismatched hybridization as follows: (i) determine the intensity ofsignals from mutant/polymorphic amplicons and from wild-type ampliconhybridization, as corrected for background signals, (ii) determine theratio of said signals (i.e., either ratio (a): mutant/polymorphic towild-type instensity; or ratio (b): wild-type to mutant/polymorphicinstensity); and setting three relative ranges of values for the ratios:(i) wherein the lowest range of ratio (a) indicates that the sample ishomozygous for wild-type and the lowest range of ratio (b) indicatesthat the sample is homozygous for or mutant/polymorph, (ii) a middlerange indicates heterozygous, and (iii) the highest range of ratio (a)indicates that the sample is homozygous for mutant/polymorph and thehighest range of ratio (b) indicates that the sample is homozygous forwild-type.
 46. The method of claim 45 further including the step ofgenerating single stranded DNA from the wild-type and mutant/polymorphamplicons.
 47. The method of claim 46 further including the step oflabeling one of the strands of either the wild-type or mutant/polymorphamplicons.
 48. The method of claim 45 wherein ratio (a) is interpretedsuch that: >2 indicates homozygous mutant/polymorph, <0.5 indicateshomozygous wild-type, 0.8 to 1.2 indicates heterozygous.
 49. A method ofcorrecting for false positive signals from mismatched probe-sample (orprobe-amplicon) binding, based on signals obtained from anoligonucleotide probe array designed to detect genetic mutations orpolymorphisms through hybridization of probes to samples, or toamplicons generated from samples, comprising: forming an array ofprobes; placing the array and the samples, or the array and theamplicons, in contact under annealing temperature and conditions;heating from the annealing temperature through a plurality of settemperature points, each said point representing the temperature atwhich a particular mismatched hybrid is expected to de-anneal;monitoring signals from the array during heating to determine thenumbers (or relative numbers) of hybrids from the start and at settemperature points; and interpreting the results from the monitoringstep based on the assumption that none of the signal at the differentset points is from mismatched hybrids which were expected to havede-annealed below said respective set points.
 50. The method of claim 49wherein the signals are from labels associated with amplicons orsamples.
 51. The method of claim 50 wherein the labels can be opticallydetected.
 52. The method of claim 49 wherein the sample or amplicons inthe mismatched hybrids differ in sequence by one nucleotide from theproperly matched sample or amplicon.
 53. The method of claim 49 whereinthe temperature ranges from 45 to 60° C.
 54. A method of designing aprobe array for use in hybridization analysis with complementaryamplicons, for detecting known mutations and polymorphisms in a genomicregion, comprising: (i) providing a family of amplicons in which onestrand is designated sense and the complementary strand is designatedanti-sense, said amplicons amplified from particular genomic regions inwhich said mutations or polymorphisms are located; (ii) selecting anamplicon from said family; (iii) aligning the selected amplicon with theremaining amplicons in the family by pairwise alignment or by multiplesequence alignment and determining homology scores with respect to theselected amplicon; (iv) ranking the amplicons in the family in order ofincreasing or decreasing homology score; (v) removing amplicons from thefamily whose homology scores exceed a preset acceptance level andplacing the removed amplicons (and the complementary probes) in aseparate group, and repeating steps (i) to (v) with another amplicon inthe family; (vi) placing each amplicon in turn in contact with theprobes in a particular group, and determining cross-hybridization withother probes in that group; (vii) selecting the complementary strand ofany probes and amplicons for which the cross-hybridization in step (vi)exceeded a preset acceptance level, and again determining thecross-hybridization With other probes in that group; and (viii) placingany probes and amplicons into a separate group where thecross-hybridization determined in step (vii) exceeds a preset acceptancelevel.
 55. The method of claim 54 wherein following step (viii) theprocess is repeated, but the objective is to generate the minimal numberof separate groups of amplicons and probes.
 56. The method of claim 54wherein following step (viii), groups with more than a predeterminednumber of amplicons (and probes) are examined and, for such groups,there is a determination whether amplicons (and probes) therein shouldbe placed into a new and separate group based on defining a new lowermaximum predetermined homology score.
 57. The method of claim 54 whereincross-hybridization is reduced by generating amplicons which are shorterthan the amplicons displaying excessive cross-hybridization.
 58. Amethod of correcting an assay image of an array of signals generatedfrom a multiplexed hybridization-mediated assay, where individualsignals indicate hybridization events, and where optically encodedcarriers are used for encoding of the individual hybridization events inthe assay, comprising: constructing a background map using signals fromnegative control carriers (i.e., encoded carriers which are notassociated with a hybridization event); and subtracting the backgroundmap signals from the assay image to produce a corrected assay image. 59.The method of claim 58 wherein the constant (i.e., the spatiallynon-varying) portion of the background map is subtracted from the assayimage, which is then divided by the corrected background map.
 60. Themethod of claim 58 wherein background map is generated by locating thecentroids of the negative control carriers included in the array at apreselected abundance, said negative control carriers being encoded andbeing designed so as to not participate in hybridization; andconstructing the associated Voronoi tessellation consisting of a seriesof polygons each containing a negative control carrier, and filling eachpolygon with the intensity of its constituent negative control carrierto produce a map.
 61. The method of claim 58 wherein filteringoperations are applied to correct for effects from neighboring negativecontrol carriers.